Project Summary A fundamental question in Molecular and Cellular Biology (Area of Science #7) is how the differentiation potential of cells is regulated. Early embryonic cells are considered pluripotent because they can differentiate into all cell types of the body. However, most of the studies aimed at understanding pluripotency have been performed in vitro, using cultured Embryonic Stem (ES) cells. Few studies have addressed the question of the significance of pluripotency in vivo. We have made progress towards answering this question. Data from our laboratory indicate that there is a global maintenance of the transcriptional program for ES cell pluripotency in the embryonic germline in vivo. Little is known about how the germline is distinguished from somatic tissues. We will test the hypothesis that the pluripotency program is essential to repress somatic differentiation in the germline. Understanding how this program represses somatic differentiation will put us in a position to harness pluripotency for novel cell-replacement therapies for degenerative diseases. A major roadblock to the use of pluripotent stem cells in the clinic is that they give rise to germ cell tumors when injected into animals. Such tumors arise spontaneously from transformation of the germline. If the pluripotency program is central to germline identity, there must be mechanisms that keep this program under control to prevent germ cell tumorigenesis. We propose to identify these mechanisms, based on insights provided by our current data. If this research is successful, we will have uncovered routes towards preventing ES cell-induced tumorigenesis and potentially reversing the course of testicular cancer. To accomplish these goals, we are developing innovative methods for rapid germline-specific and drug-inducible genetic manipulations in the mouse. Our trajectory is aimed at deciphering the biological significance and molecular regulation of pluripotency, towards making the use of pluripotent stem cells in regenerative medicine a reality.